CN113181118A - Preparation for treating bladder cancer - Google Patents

Abstract

Compositions and methods for making and using proliposomes and liposomal formulations of chemotherapeutic agents are disclosed. Proliposome and liposome formulations of chemotherapeutic agents, as well as medicaments and dosage forms comprising such formulations, may be used with treatment regimens for bladder cancer and urothelial cancer. Thus, the formulations, medicaments and dosage forms of the invention are suitable for the treatment of bladder cancer and for the treatment of urothelial cancer by intravesical administration. The formulations according to the invention comprise (a) a taxane (e.g. paclitaxel, docetaxel) or cisplatin, (b) a first phospholipid, Dipalmitoylphosphatidylcholine (DMPC) and (c) a second phospholipid, dimyristoylphosphatidylglycerol sodium (DMPG). The proliposomal formulation forms liposomes when contacted with an aqueous carrier.

Description

Preparation for treating bladder cancer

Divisional application

The application is a divisional application with application number 201780002340.X, application date 2017, 09.01 and titled "preparation for treating bladder cancer".

Cross Reference to Related Applications

This application claims priority to U.S. application nos. 62/275,941 and 62/275,936, both filed on 2016, 1, 7, and U.S. application No. 62/421,137, filed on 2016, 11.

Technical Field

The invention described herein relates to proliposomes (proliposomal) and liposomal formulations of therapeutic agents, and their use in the treatment of bladder cancer.

Background

Administration of chemotherapeutic agents for the treatment of bladder cancer typically involves intravesical administration of the agent directly into the bladder using a urinary catheter. However, this method of administering chemotherapeutic agents is for use with chemotherapeutic agents, such as paclitaxel

Disorders are provided for the treatment of bladder cancer (Hadaschik et al, "Patlitaxel and cistatin as intragenic agents against non-human-innovative lens scanner" BJUI 101: 1347-. More specifically, paclitaxel precipitates, for example, in a pH environment within the bladder, where the pH may range from 4.5 to 8, and thus no longer has bioavailability. Although paclitaxel may be dissolved in dimethyl sulfoxide (DMSO), the amount of DMSO required to maintain an effective dose in solution for the treatment of bladder cancer is not pharmaceutically acceptable. Thus, there is a need to formulate stable formulations of chemotherapeutic agents that can be administered intravesically without precipitating in the bladder. This need is met by the compositions and methods described herein, which formulate therapeutic doses of chemotherapeutic agents as free-flowing proliposomal powder dispersions that can be dispersed in aqueous media over a wide range of pH values without causing drug precipitation.

Disclosure of Invention

The present invention relates to compositions and methods for the preparation and use of proliposomes and liposomal formulations of chemotherapeutic agents. In various aspects, the compositions of the invention are precursor liposome powder dispersions comprising (a) a taxane or cisplatin as a chemotherapeutic agent, (b) Dipalmitoylphosphatidylcholine (DMPC) and (c) dimyristoylphosphatidylglycerol sodium (DMPG). The weight ratio of a to b to c is 1 (1.3-4.5) to 0.4-2.5.

In some aspects of the invention, the chemotherapeutic agent in the proliposome powder dispersion is a taxane. Examples of taxanes useful in preparing the formulations of the present invention include, but are not limited to, paclitaxel, docetaxel, cabazitaxel, tesetaxel, DJ-927, TPI 287, larotaxel, otaxel, DHA-paclitaxel, or combinations thereof. For example, the taxane may be (a) docetaxel and the weight ratio of a: b: c is 1 (1.3-2.0) to (0.4-2.0).

In other aspects, the chemotherapeutic agent is cisplatin. In addition to (a) cisplatin, (b) DMPC, and (c) DMPG, the proliposome powder dispersion according to the present invention may further comprise (d) cholesterol and have a weight ratio a: b: c: d of 1 (2.5-4.5) to (1.0-2.5) to (0.5-1).

In various aspects of the invention, the proliposome powder dispersion may include (a) paclitaxel, (b) DMPC, and (c) DMPG in a weight ratio a: b: c of 1 (1.3-3.8) to (0.4-1.5). In addition to (a) taxane or cisplatin, (b) DMPC, and (c) DMPG, the formulations of the present invention may include (d) cholesterol and have an a: b: c: d weight ratio of 1 (1.3-3.8) to (0.4-1.5) to (0.5-1).

In some aspects, the invention relates to a pharmaceutical composition comprising any of the proliposomal powder dispersions of the invention and at least one pharmaceutically acceptable excipient. In other aspects, the invention relates to a dosage form comprising any of the pharmaceutical compositions.

In other aspects, the invention relates to methods of preparing liposomal formulations of taxanes or cisplatin. Liposome formulations can be prepared by hydrating (hydrating) any of the proliposomal powder dispersions of the invention in an aqueous carrier. The formulations of the present invention may also be prepared by: stirring (sturing), mixing and/or homogenizing the first lipid and the second lipid to disperse in the aqueous carrier to form a dispersion; adding a taxane or cisplatin to the dispersion of the first lipid and the second lipid; homogenizing a dispersion of a first lipid, a second lipid and taxane or cisplatin to obtain taxane-or cisplatin-incorporated liposomes; homogenizing the liposomes to obtain nanoscale liposome particles in dispersion; and adding a cryoprotectant/lyoprotectant. In some aspects of the invention, the dispersion may be lyophilized to form a proliposome powder dispersion. In other aspects, the homogenization step may be performed at elevated pressure and/or at a temperature above the Tc/Tg of the lipid.

In some aspects of the invention, the invention relates to a pharmaceutical composition comprising any of the liposomal formulations of the invention.

The invention also relates to methods of treating bladder cancer in a patient by administering to the patient the pharmaceutical compositions of the invention. In some aspects, the pharmaceutical composition can be administered by intravesical delivery, and the cancer is a non-muscle invasive bladder cancer. In certain aspects of the invention, the taxane or cisplatin is still soluble in the bladder at any pH of 4.5 to 8.

The invention further relates to a method of treating upper urinary tract epithelial cancer in a patient by administering to the patient a pharmaceutical composition of the invention. In some aspects, the pharmaceutical composition can be administered into the ureter and/or renal pelvis in order to treat upper urothelial cancer.

Drawings

Fig. 1 shows a photomicrograph under a light microscope of paclitaxel-incorporated liposomes prepared using the method for preparing liposomes according to example 6 (bars indicate 100 μm).

FIG. 2 shows a graph of animal body weight at day 0, day 7 and day 14 of treatment with 10mg/kg proliposomal intravesical paclitaxel formulation (PLIP-001, referred to as TSD-001 in FIG. 2), 15mg/kg PLIP-001, 15mg/kg abraxane or saline, as discussed in example 8.

FIG. 3 depicts the average body weight of the animals at day 14 after administration of 10mg/kg PLIP-001(PLIP-001 is referred to as TSD-001 in FIG. 3), 15mg/kg PLIP-001, 15mg/kg abraxane, or saline.

FIG. 4 shows the average bladder weights of animals at day 14 after administration of 10mg/kg PLIP-001(PLIP-001 is referred to as TSD-001 in FIG. 4), 15mg/kg PLIP-001, 15mg/kg abraxane, or saline.

FIG. 5 shows the average tumor area at day 14 after animals were administered 10mg/kg PLIP-001(PLIP-001 is referred to as TSD-001 in FIG. 5), 15mg/kg PLIP-001, 15mg/kg abraxane or saline.

FIG. 6 shows the mean tumor area measured histologically on day 14 after animals were administered 10mg/kg PLIP-001, 15mg/kg PLIP-001, 15mg/kg abraxane or saline.

FIG. 7 shows the mean tumor area on day 21 after 21 days of animal administration of 0.5mg/kg PLIP-001(PLIP-001 is referred to as TSD-001 in FIG. 7), 2.5mg/kg PLIP-001, 5mg/kg PLIP-001, 5mg/kg paclitaxel (pure paclitaxel dissolved in DMSO), or saline.

FIG. 8 shows plasma paclitaxel levels at day 21 after intravesical administration of 0.5mg/kg PLIP-001, 2.5mg/kg PLIP-001, 5mg/kg PLIP-001, and unformulated paclitaxel under 21 days of treatment.

Figure 9 shows paclitaxel concentrations in tissues from cryosections following PLIP-001 and Abraxane administration to isolated male porcine bladder (ex vivo).

Detailed Description

The present invention relates to compositions and methods for the preparation and use of proliposomes and liposomal formulations of chemotherapeutic agents. The formulations of the present invention, as well as medicaments and dosage forms comprising such formulations, may be used in conjunction with a treatment regimen for bladder cancer. The formulations, medicaments and dosage forms of the invention are suitable for administration of chemotherapeutic agents to the bladder as well as the ureters and renal pelvis. The formulations, medicaments, and dosage forms of the invention can prevent precipitation of the formulated chemotherapeutic agent in an aqueous urine environment at pH levels typical for the bladder environment, which can range from 4.5 to 8.

Various types of bladder cancer, including non-muscle invasive bladder cancer (NMIBC), are treated by the compositions and methods of the present invention. The proliposomes and liposome formulations of the invention may be used to treat urothelial cancer, also known as transitional cell carcinoma. Urothelial cancer is the most common type of bladder cancer, accounting for approximately 90% of all bladder cancers. These cancers are usually superficial in about 75% of cases where they do not enter the deeper layers of the bladder wall. The formulations of the invention may also be used to treat other types of bladder cancer, such as squamous cell carcinoma or adenocarcinoma.

Most superficial tumors (i.e., those tumors localized to the bladder mucosa and lamina propria) are treated by urologists through cystoscopy surgery and selective intravesical drug therapy. Although these superficial bladder cancers frequently recur and may be multifocal, survival after treatment is often excellent. However, if the cancer has penetrated the muscle wall of the bladder (i.e., the cancer has progressed to a muscle-invasive bladder cancer that invades deeper into the bladder wall, and may be close to organs such as the uterus, vagina, or prostate), the prognosis is generally worse. Approximately 50% of patients with muscle-invasive bladder cancer will develop metastatic disease. Thus, there is a clear need for effective treatments for bladder cancer.

Proliposome and liposome preparation

The method of the invention for treating bladder cancer comprises administering a liposomal suspension comprising poorly water-soluble drug-incorporated liposomes. The liposomes can be nanoscale liposomes. The liposomes incorporate a chemotherapeutic agent or a combination of chemotherapeutic agents. Liposomes can be prepared by hydrating a powder dispersion of the proliposomes of the invention. The proliposome powder dispersion is a dry powder that can be formed as known in the art, for example, by a cast film process, such as described in examples 1-4 below and in U.S. patent nos. 9,445,995 and 6,759,058, the entire contents of which are incorporated herein. The liposome preparation can be prepared by dispersing a proliposome powder dispersion in an aqueous carrier.

The liposome formulation can also be prepared by an organic solvent free process as described in example 6 below. Typically, the first lipid and the second lipid may be dispersed in the aqueous carrier by stirring, mixing and/or homogenization to form a dispersion. The taxane or cisplatin may then be added to the dispersion of the first lipid and the second lipid, and the dispersion of the first lipid, the second lipid, and the taxane or cisplatin may be homogenized to obtain taxane or cisplatin incorporated liposomes. The liposomes can be further homogenized to obtain nanoscale liposome particles in dispersion. Lyoprotectants/cryoprotectants may be added to the dispersion. If desired, the dispersion can be lyophilized to obtain a proliposomal powder dispersion of taxane or cisplatin. More generally, this method can be used in combination with any lipid or phospholipid to form a preparation of a poorly water soluble drug (e.g., a taxane or cisplatin). Examples of suitable phospholipids that may be used in the process of preparing the formulations of the invention include Distearoylphosphatidylcholine (DSPC), Dipalmitoylphosphatidylcholine (DPSC), Dimyristoylphosphatidylcholine (DMPC), egg phosphatidylcholine (eg-PC), soy phosphatidylcholine (soy-PC), dimyristoylphosphatidylglycerol sodium (DMPG), 1, 2-dimyristoyl-phosphatidic acid (DMPA), Dipalmitoylphosphatidylglycerol (DPPG), dipalmitoyl phosphate (DPP), 1, 2-distearoyl-sn-glycero-3-phospho-rac-glycerol (DSPG), 1, 2-distearoyl-sn-glycero-3-phosphatidic acid (DSGPA), Phosphatidylserine (PS) and Sphingomyelin (SM), or a combination of any of the foregoing phospholipids.

The proliposome powder dispersions and liposomes of the present invention comprise a phospholipid component comprising a first phospholipid dimyristoyl phosphatidylcholine (DMPC) and a second phospholipid dimyristoyl phosphatidylglycerol sodium (DMPG).

The proliposome powder dispersion of the present invention comprises at least (a) a chemotherapeutic agent, (b) a first phospholipid DMPC and (c) a second phospholipid DMPG, which are dispersed in each other and form liposomes when contacted with an aqueous solution. For example, a proliposome powder dispersion can comprise a weight/weight ratio of (a), (b), and (c) in the range of (1.0): (1.0-4.5): (0.4-2.5). The proliposome powder dispersion may further contain (d) cholesterol in addition to the components (a) to (c). Thus, the proliposomal preparation may comprise (a), (b), (c), (d) in a weight/weight ratio of (a) to (b) to (c) to (d) in the range of (1.0) to (1.0-4.5) to (0.1-2.5) to (0.1-2.0).

When phospholipids such as DMPC and DMPG are placed in an aqueous environment, the hydrophilic heads are clustered together in a linear configuration with their hydrophobic tails aligned substantially parallel to each other. The second row of molecules is then aligned end-to-end with the first row because the hydrophobic end tries to avoid the aqueous environment. To avoid contact with the aqueous environment to the greatest extent, i.e., at the edges of the bilayer, while minimizing the surface area to volume ratio, and thereby achieving the smallest energy conformation, two rows of phospholipids, called phospholipid bilayers or laminae, are pooled into the liposome. In doing so, the liposomes (or phospholipid spheres) entrap some of the aqueous medium, and there may be any substance dissolved or suspended in the core of the spheres. This includes various components of the proliposome powder dispersions of the invention, such as chemotherapeutic agents.

According to the methods of the invention, prior to administration of one or more chemotherapeutic agents, typically by intravesical delivery into the bladder, the powder dispersion of proliposomes comprising the chemotherapeutic agent is hydrated in water or another pharmaceutically acceptable aqueous carrier (e.g., saline) such that liposomes are formed, encapsulating the chemotherapeutic agent within the liposomes. In addition to water or aqueous carriers, the resulting liposome suspension may contain lyoprotectants, such as mannitol, sucrose or trehalose. Typically, the w/w ratio of the lyoprotectant/cryoprotectant component to the drug component (lyoprotectant/cryoprotectant: drug) of the liposomal formulation is about (0.5:1.0) to (5.5: 1.0). For example, a liposome suspension for use in a method of treatment according to the present invention may be prepared by mixing a precursor liposome powder dispersion comprising (1.0): 1.0-4.5): 0.1-2.5): 0.5-5.5) of (a) a chemotherapeutic agent, (b) DMPC and (c) DMPG, and (e) a lyoprotectant/cryoprotectant.

The proliposomes and liposome formulations of the invention can be adapted to a variety of chemotherapeutic agents known in the art for the treatment of bladder cancer. The present invention includes, but is not limited to, taxanes including paclitaxel, docetaxel, DJ-927, TPI 287, larotaxel, otaxel, DHA-paclitaxel, cabazitaxel and tesetaxel, cisplatin or mixtures thereof, and combinations with other chemotherapeutic agents.

For example, a proliposomal powder dispersion of the invention comprising a taxane derivative drug ("liposomal intravesical taxane (PLIT) formulation") may comprise (a) a taxane, (b) a first phospholipid DMPC, and (c) a second phospholipid DMPG. The proliposome powder dispersion may comprise (a), (b), (c) in a weight/weight ratio selected from (1.0): 1.0-3.8): 0.2-1.5) or any ratio therein of (a): (b): (c). For example, in the proliposome dispersion of the present invention, the weight/weight ratio of paclitaxel to DMPC to DMPG of (a) to (b) to (c) may be (1.0) to (3.15) to (1.00), respectively; or (1.0) to (3.20) to (1.05); or (1.0) to (3.25) to (1.10); or (1.0) to (1.43) to (0.567) ratio or any ratio contained therein. The proliposome powder dispersion of the invention can consist essentially of (a) a taxane, (b) DMPC, and (c) DMPG in any of the weight/weight ratios shown, or it can consist of those components in any of those ratios.

In addition to the taxane, DMPC, and DMPG, the proliposome powder dispersions described herein can also comprise (d) cholesterol. Thus, the proliposome powder dispersion according to the invention may comprise a weight/weight ratio of (a) to (b) to (c) to (d) selected from (1.0) to (1.0-3.8) to (0.4-1.5) to (0.5-1) or any ratio comprised therein. For example, the proliposome powder dispersion of the invention may comprise (a) paclitaxel, a first phospholipid, (b) DMPC, a second phospholipid, (c) DMPG, and (d) cholesterol, wherein the weight/weight ratio of (a) to (b) to (c) to (d) is (1.0) to (3.40) to (1.25) to (0.70); or (1.0) to (3.45) to (1.30) to (0.75); or (1.0) to (3.50) to (1.35) to (0.80); or any ratio contained therein. The proliposome powder dispersion of the invention can consist essentially of (a) a taxane, (b) DMPC, (c) DMPG, and (d) cholesterol in any of the weight/weight ratios indicated, or it can consist of those components in any of those ratios.

Alternatively, the proliposomes and liposome formulations of the invention may, for example, comprise cis-diamminedichloroplatinum (II), commonly known as cisplatin, as the chemotherapeutic agent. A proliposomal powder dispersion of the invention comprising cisplatin ("proliposomal intravesical cisplatin (PLIC) preparation") may comprise (a) cisplatin, (b) a first phospholipid DMPC, and (c) a second phospholipid DMPG. The cisplatin proliposome powder dispersion may comprise (a), (b), (c) in a weight/weight ratio selected from (1.0): (2.5-4.5): (1-2.5) or any ratio therein (a): (b): (c). For example, the weight/weight ratio of (a): (b): (c) may be (1.0): (2.7): (1.2); or (1.0) to (2.75) to (1.21); or (1.0) to (2.76) to (1.22); or (1.0) to (2.77) to (1.2); or (1.0) to (2.78) to (1.22); or any ratio contained therein. In (a) a proliposome powder dispersion of cisplatin, the weight/weight ratio of (a) to (b) to (c) may be (1.0) to (2.7) to (1.2); or (1.0) to (2.75) to (1.21); or (1.0) to (2.76) to (1.22); or (1.0) to (2.77) to (1.2); or (1.0) to (2.78) to (1.22); or any ratio contained therein. The proliposomal powder dispersion of the invention can consist essentially of (a) cisplatin, (b) DMPC, and (c) DMPG in any of the weight/weight ratios shown, or it can consist of those components in any of those ratios.

In the proliposome powder dispersion according to the invention, the weight/weight ratio of (a) to (b) to (c) may be (1.0) to (4.1) to (2.1); or (1.0) to (4.15) to (2.25); or (1.0) to (4.16) to (2.26); or (1.0) to (4.17) to (2.27); or any ratio therein. In a proliposomal dispersion wherein (a) is cisplatin, the first phospholipid (b) is DMPC and the second phospholipid (c) is DMPG, the weight/weight ratio of (a) to (b) to (c) can be (1.0) to (4.1) to (2.1); or (1.0) to (4.15) to (2.25); or (1.0) to (4.16) to (2.26); or (1.0) to (4.17) to (2.27); or any ratio contained therein. The proliposomal powder dispersion of the invention can consist essentially of (a) cisplatin, (b) DMPC, and (c) DMPG in any of the weight/weight ratios indicated, or it can consist of those components in any of those ratios.

In addition to (a) cisplatin, (b) DMPC and (c) DMPG, the precursor liposome powder dispersion of cisplatin may comprise (d) cholesterol. Such cisplatin preparations may comprise a weight/weight ratio of (a) to (b) to (c) to (d) selected from (1.0) to (2.5-4.5) to (1.0-2.5) to (0.5-1) or any ratio comprised therein. (a) The weight/weight ratio of (b) to (c) to (d) may be, for example, (1.0) to (2.7) to (1.2) to (0.6); or (1.0) to (2.75) to (1.21) to (0.65); or (1.0) to (2.76) to (1.22) to (0.7); or (1.0) to (2.77) to (1.2) to (0.75); or (1.0): 2.78): 1.22): 0.8; or (1.0): 2.78): 1.22): 0.9; or any ratio contained therein.

The liposomal powder dispersions and liposomal formulations of the present invention can be used in pharmaceutical formulations or dosage forms that are administered to individuals in need of chemotherapeutic agents (e.g., paclitaxel, docetaxel, cisplatin, etc.). The pharmaceutical formulation or dosage form according to the invention may be administered for the treatment of bladder cancer. More particularly, in therapeutic applications, the pharmaceutical formulation or dosage form is administered to an individual already having bladder cancer in an amount sufficient to at least one of remove all symptoms or at least partially alleviate the symptoms of bladder cancer. The dose of chemotherapeutic agent effective for this use will depend on the stage, severity and course of the bladder cancer, previous treatments, the health of the individual, body weight, response to the drug and/or the judgment of the treating physician.

To prepare a precursor liposome powder dispersion of a chemotherapeutic agent, as described in examples 1-4 below, the chemotherapeutic agent (e.g., paclitaxel) can be dissolved in ethanol along with the lipids, and the film can be cast using a rotary flash evaporator. The dried film may be hydrated using physiological saline or water or any other pharmaceutically acceptable aqueous carrier. This provides a liposome dispersion. The liposome dispersion is then extruded using an Emulsiflex TM-C5(Avestin, Canada) or similar high pressure homogenizer or suitable apparatus known in the art to achieve the desired particle size. In the liposomes of the present invention, the particles may be of the nanoscale. Liposomes of the invention may generally have a particle size of up to 700nm, up to 500nm, up to 250nm, up to 200nm or up to 100 nm.

For the liposome dispersions of the present invention, suitable excipients can be added externally and lyophilized to obtain a precursor liposome powder dispersion, i.e., the excipients are added "externally". For example, the proliposomal powder dispersion according to the invention can be mixed with at least one pharmaceutically acceptable excipient. Exemplary pharmaceutically acceptable excipients include, but are not limited to: (a) cryoprotectants, fillers or extenders, such as mannitol, starch, lactose (e.g., lactose monohydrate), sucrose, glucose, trehalose and silicic acid; (b) binders, for example, cellulose derivatives, including hydroxypropylmethylcellulose (which is commercially available as benecel (tm)), hydroxypropylcellulose (which is commercially available as klucel (tm) (Ashland Inc-Covington, KY)), starch, alginates, gelatin, polyvinylpyrrolidone, sucrose and gum arabic, (c) absorption enhancers, such as quaternary ammonium compounds.

Intravesical delivery

The formulations and dosage forms of the invention can be used to intravesically deliver therapeutic doses of chemotherapeutic agents (e.g., taxanes, such as paclitaxel, docetaxel and/or cisplatin) to the bladder. Intravesical treatment involves the infusion of a therapeutic agent directly into the bladder by insertion of a urethral catheter. In a typical version of intravesical infusion, sterile catheterization may be performed with a straight catheter or a coude catheter. The bladder was emptied completely. A catheter tip syringe containing a therapeutic adapter may be inserted at the tip of the syringe to prevent spillage or splash during insertion. Alternatively, a painted tube attached to a vial may be inserted into the catheter and infused with the chemotherapeutic agent by gravity flow or gentle injection. The patient's pain can be assessed. The syringe or vial can be removed intact with the tube. The catheter is squeezed closed and the catheter or plugged catheter is removed as indicated, using sterile gauze to help absorb any drops. If the patient has difficulty maintaining the solution, a Foley catheter may be used, and a catheter plug may be inserted into the end of the catheter after infusion, leaving the chemotherapeutic agent in the bladder for a prescribed amount of time, typically one to two hours. Depending on the mobility of the patient, the catheter may be withdrawn at the end of the desired dwell time, or the patient may be connected to a urine drainage bag to drain the chemotherapeutic agent. Once the catheter is properly removed and discarded, the perineal region is examined for leaks and the patient reevaluated for pain. The patient is instructed to attempt to remain on treatment for 1 to 2 hours. Historically, the patient was instructed to lie down and reposition from the left to the right every 15 minutes, then move the bubble back out of the catheter and ensure that the drug was in contact with all areas of the bladder.

Embodiments of intravesical drug delivery devices and methods for deploying those devices into the bladder are described in the following U.S. patent application publications: u.s.20150165178; U.S. 2012/0203203; U.S. 2012/0089122; U.S. 2012/0089121; u.s.2011/0218488; u.s.2011/0202036; u.s.2011/0152839; u.s.2011/0060309; U.S. 2010/0331770; U.S. 2010/0330149; U.S. 2010/0003297; U.S. 2009/0149833; and u.s.2007/0202151, all of which are incorporated herein in their entirety.

In addition to intravesical delivery, the formulations and dosage forms of the invention can be administered into the ureter and/or renal pelvis using suitable catheter devices and protocols known in the art. Such delivery of chemotherapeutic agents may be useful for treating, for example, epithelial cancers.

When the formulations and dosage forms of the present invention are delivered from a drug delivery device, the formulations and dosage forms may be encapsulated in the device in a variety of forms depending on the particular mechanism by which the device releases the proliposomal powder dispersion, liposomal formulation, pharmaceutical formulation and dosage form into the urine within the bladder and/or other parts of the renal system. The dosage form may be a solid, semi-solid, or other non-liquid form (e.g., a powder or compressed powder), which advantageously may facilitate stable storage of the chemotherapeutic agent prior to use of the device, and which advantageously may allow storage of the chemotherapeutic agent in a volume that is smaller than would be possible if the agent were encapsulated in a liquid solution or suspension.

When using the formulations of the present invention, the chemotherapeutic agent may remain soluble in human urine at typical urine pH of 4.5-8 after intravesical delivery. Moreover, the formulation of the present invention allows the chemotherapeutic agent to adhere to the bladder wall, and the chemotherapeutic agent can last up to 3 days in the excreted urine.

Parenteral administration

The proliposomal powder dispersions, liposomal formulations, pharmaceutical formulations and dosage forms of the invention can be used to prepare compositions for the parenteral delivery of therapeutic doses of a taxane (e.g., paclitaxel or docetaxel) or cisplatin to a patient. Parenteral administration includes intravenous, intra-arterial, intramuscular, intracerebroventricular, or subcutaneous routes of administration.

Injectable compositions may be prepared in conventional forms as liquid solutions or suspensions, solid forms suitable for dissolution or suspension in a liquid prior to injection, or as emulsions. Injections, solutions and emulsions may also comprise one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. In addition, if desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubilizing agents and other such agents, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.

Suitable pharmaceutical excipients known in the art may be combined with the proliposomal powder dispersion according to the invention to produce a pharmaceutical formulation or dosage form.

Combination therapy

The liposomal powder dispersion, liposomal formulation, pharmaceutical formulation, and dosage form according to the invention can be administered in combination with other therapeutic agents that reduce the severity or eliminate side effects associated with chemotherapy, including nausea, vomiting, loss of appetite, diarrhea, loss of taste, hair loss that may occur, numbness/tingling/cold/bluish discoloration of the hands and feet, arm or leg pain/redness/swelling, loss of reflexes, loss of balance, difficulty walking, muscle spasm/twitching/weakness, neck or back pain, oral or tongue pain, joint pain, swelling of the legs or feet, mental/mood changes, head pain, rapid/irregular heartbeat, hematuria, vomit that looks like coffee grounds, black blood or bloody stool, pain or difficulty in urination, pain or difficulty in the waist or side, or vision changes (e.g., blurred vision, difficulty in seeing color).

In certain cases, it is suitable to administer the proliposomal powder dispersions, pharmaceutical preparations and dosage forms according to the invention together with another therapeutic agent. For example, the paclitaxel proliposome powder dispersion may be used in the form of a pharmaceutical formulation or dosage form that is administered as part of a combination therapy that includes gemcitabine for the treatment of bladder cancer. The cisplatin precursor liposome powder dispersion according to the present invention can be used in pharmaceutical preparations or dosage forms administered as part of a combination therapy comprising 5-fluorouracil (5-FU) for the treatment of bladder cancer. The paclitaxel proliposome powder dispersion may also be used in pharmaceutical preparations or dosage forms administered as part of a combination therapy that includes a proliposomal cisplatin preparation.

In the case of a combination therapy, the other agents need not be administered in the same pharmaceutical composition and may be administered by different routes due to different physical and chemical properties. For example, initial administration may be performed according to a determined protocol, and then based on the observed effect, the dosage, mode of administration, and number of administrations may be further modified.

Depending on the stage and type of cancer, the condition of the patient, and the actual choice of compound used, the multiple therapeutic agents may be administered concurrently (e.g., simultaneously, substantially simultaneously, or within the same treatment regimen) or sequentially. Determining the order of administration and the number of repeated administrations of each therapeutic agent during a treatment regimen can be based on the assessment of the disease being treated and the condition of the individual.

The individual chemotherapeutic agents of such combinations are administered sequentially or simultaneously in separate or combined pharmaceutical formulations. For example, the single therapeutic agent(s) may be administered simultaneously in a combined pharmaceutical formulation. One skilled in the art will appreciate the appropriate dosages of known therapeutic agents.

The combination according to the invention may conveniently be presented for use in the form of a pharmaceutical composition together with a pharmaceutically acceptable diluent or carrier.

Examples

Examples 1-4 below describe the preparation of proliposomal intravesical paclitaxel (PLIP) formulations PLIP-003, PLIP-006, PLIP-021 and PLIP-023, respectively. The PLIP formulation described above was prepared by: all the drug and lipid components of each formulation as described in tables 1-4, respectively, were dissolved together in 10mL ethanol in a 500mL round bottom flask by placing in a water bath at 50 ℃. Films were cast from the lipid component and ethanol mixture by drying under reduced pressure using a rotary flash evaporator (Buchi). The membrane was completely dried at room temperature and under reduced pressure (150 to 200 mbar) overnight. The membrane was hydrated by placing the flask in a water bath at 50 ℃ using 20mL of saline. The flask was rotated using a Buchi rotary flash evaporator resulting in the formation of a liposome dispersion. Then using Nano at room temperature under high pressure

The high pressure homogenizer homogenizes the dispersion to produce unilamellar liposomes having particles in the size range of 100-200 nm. Then use

The homogenizer extrudes the prepared dispersion. Extrusion was carried out using a polycarbonate film with a pore size decreasing from 1 μm to 0.2. mu.m. At the final extrusion, mannitol was added in the amounts as described in tables 1 to 4, respectively, and the mixture was lyophilized to obtain a proliposome powder dispersion.

Example 1. PLIP-003.

TABLE 1

Composition (I)	Measurement of
		Paclitaxel (mw 853.9Da)	24mg
DMPG(mw＝688.9Da；Tc＝23℃)	25.2mg
		DMPC(mw＝677.9Da；Tc＝24℃)	77.5mg
Mannitol	100mg

Example 2 PLIP-006

TABLE 2

Composition (I)	Measurement of
		Paclitaxel (mw 853.9Da)	25.2mg
DMPG(mw＝688.9Da；Tc＝23℃)	33.8mg
		DMPC(mw＝677.9Da；Tc＝24℃)	84.4mg
Cholesterol (mw 386.65Da)	20.1mg
		Mannitol	27mg

Example 3 PLIP-021

TABLE 3

Composition (I)	Measurement of
		Paclitaxel	27.4mg
DMPG	12.2mg
		DMPC	90.4mg
Mannitol	50mg

Example 4 PLIP-023

TABLE 4

Composition (I)	Measurement of
		Paclitaxel	25.2mg
DMPG	18.2mg
		DMPC	90.4mg
Mannitol	50mg

Example 5 in vitro analysis of the efficiency of PLIP-003, PLIP-006, PLIP-021 and PLIP-023. Determination of paclitaxel formulations PLIP-003, PLIP-006, PLIP-021 and PLIP-023 human urothelial cancer cell line T24 (using a sulforhodamine B (SRB) -based assay

HTB-4TM)、5637(

HTB-9TM) and HT-1376(

CRL-1472TM) Inhibitory Concentration (IC) 50. For use in the assay, the paclitaxel formulation was redispersed in physiological saline to a concentration of 2-5mg/mL paclitaxel. The dispersed formulation formed a clear solution. Pure unformulated paclitaxel solution ([6 mg/100. mu.L)]In DMSO) was used as a control formulation.

Cells were treated at 5X 10³The density of individual cells/well was plated onto 96-well plates and incubated at 37 ℃ and 5%CO₂And culturing for 24 hours. The dispersed paclitaxel formulation and the pure drug control were added to the medium of the plated cell cultures. After a period of 72 hours of treatment with the formulation, the medium was aspirated. Treated cells were fixed by slowly adding 100. mu.l of 10% trichloroacetic acid (TCA) to each well and the plates were incubated at 4 ℃ for at least 1 hour. After incubation, the plates were washed five times with tap water without flowing water directly into the wells, the plates were air dried at room temperature, and 50 μ Ι of 0.4% w/v SRB (in 1% acetic acid) was added to each well. Plates were incubated in SRB solution for 20 to 30 minutes at room temperature. After that, the plate was washed 5 times with 1% acetic acid and air-dried at room temperature. Protein-bound SRB was detected by adding 100. mu.l of 10mM Tris base solution to each well and allowing the Tris solution to dissolve the SRB for 5 to 10 minutes. The plates were read using a microplate reader at an absorbance of 565 nm. Table 5 reports the IC50 values for PLIP-003, PLIP-006, PLIP-021, PLIP-023 and unformulated paclitaxel.

TABLE 5

Example 6. an alternative process for preparing nano-scale poorly water-soluble drug-incorporated liposomal vesicles is performed as follows:

1. weighing the lipid components DMPC and DMPG and transferring into an aqueous medium;

2. the aqueous medium is maintained at a temperature higher than the Tc/Tg of the lipid fraction;

3. hydrating the lipid by allowing the lipid mixture to stand or by stirring, mixing and/or homogenizing;

4. adding a poorly water-soluble drug (modified taxane, such as paclitaxel, or platinum-containing drug, such as cisplatin) to the lipid dispersion, allowing the mixture of drug and lipid to continue stirring,

5. to obtain liposomes, a dispersion comprising a lipid and a drug is homogenized at high pressure and at a temperature above the Tc/Tg of the lipid. Homogenization continues until the drug is incorporated into the liposomes. Drug incorporation was confirmed by observing the liposomes under a microscope without any drug crystals;

6. once the drug is incorporated, the subsequent homogenization is carried out slightly above, at or below the Tc/Tg of the lipid, to obtain liposomal vesicles incorporating the nanoscale drug; and

7. suitable cryoprotectants/lyoprotectants are added to the liposome vesicles, followed by lyophilization to obtain pre-drug-loaded liposomes.

Advantages of the above alternative methods of preparing liposomal vesicles incorporating nanoscale drugs include the elimination of the need for organic or caustic solvents, such as ethanol, chloroform, and/or ethers. Furthermore, such methods involve a smaller number of unit operations and/or a smaller number of instruments involved in the process. Compared to the cast membrane process described in examples 1-4 (two hours preparation time compared to two days), this process also requires significantly less time to obtain drug-incorporated liposome vesicles. This is a simple, fast and economical process.

Example 7 PLIP-001. An alternative method of preparing liposome vesicles incorporating a nanoscaled poorly water soluble drug as described in example 6 was used to prepare a paclitaxel formulation PLIP-001 comprising the ingredients described in table 6. The amount of PLIP ingredient may be scaled to the amount of PTX based on the weight/weight ratio.

TABLE 6 PLIP-001

Example 8 efficacy assessment of PLIP-001 on human bladder cancer in an in situ mouse model. Paclitaxel (PTX) is highly active against metastatic bladder cancer; thus, PTX is a potential candidate for adjuvant intravesical therapy to prevent recurrence and progression of NMIBC. PTX is lipophilic. Existing preparations (e.g., paclitaxel @)

) It is insoluble in acidic water environment in bladder. The lipophilic nature of PTX, if properly formulated, creates the potential for urothelial penetration and delivery into the submucosa. The following studies demonstrate the successful delivery of PLIP-001 formulated PTX to the bladder, as well as in vitro and in vivo demonstration of the PLIP-001 concept.

The proliposomal formulations of paclitaxel were evaluated using an in situ mouse model. Bladder cancer cell lines such as KU7/GFP clone 6 were used in these studies. KU7/GFP clone 6 was stably transfected with green fluorescent protein and these cell lines were used for all in vivo studies. Watanabe et al describe the KU7/GFP clone 6 cell line. Cells were cultured in modified minimal essential medium supplemented with 10% FCS and incubated at 37 ℃ under 5% CO 2. Tumors generated from KU7/GFP cells were generated in vitro. Tumors were implanted into the bladder of female mice. Seven days after implantation of KU7/GFP tumor, mice were divided into the following four experimental treatment groups, where mice received either: 10mg paclitaxel/kg body weight (10mg/kg) administered PLIP-001 (group 1); 15mg/kg, administered PLIP-001 (group 2); 15mg/kg of Abraxane (group 3) administered as a nanoparticle albumin-bound form of paclitaxel manufactured by Celgene; or saline (group 4). The foregoing formulation and saline were administered on days 0, 7 and 14 after tumor implantation.

Tables 7, 8 and 9 show body weight and average body weight values for animals in each of groups 1-4 on days 0, 7 and 14 of treatment, respectively. Figure 2 shows body weight plots for treatment days 0, 7 and 14.

Table 7 body weight on day 0 of PLIP-001 treatment after tumor implantation.

Table 8 body weights at day 7 of PLIP-001 treatment after tumor implantation.

Table 9 body weights at day 14 of PLIP-001 treatment after tumor implantation (figure 3).

Bladder weights (B-W) of the treated mice in groups 1-4 measured on day 14 of treatment are reported in table 10. Statistical analysis of the comparison of bladder weights for each group is reported in table 6, and bladder size for the treatment group is reported in table 11.

TABLE 10 bladder weight

TABLE 11 statistical analysis

Group of	G1 to G2	Gl ratio G3	G3 to G2
				T-test	0.223	0.832	0.146
Group of	G1 to G4	G2 to G4	G3 to G4
				T-test	0.167	0.257	0.140

TABLE 12 bladder size

(in the tables, indicates that a tumor is present outside the bladder, which may occur due to bladder perforation when mice are inoculated with cancer cells. intravesical administration of chemotherapeutic drugs is not expected to have an effect on the tumor when it is located outside the bladder).

Example 9. evaluation of the efficacy of paclitaxel proliposomal formulations by measuring tumor area. KU-7-GFP human bladder cancer in situ

Model: human bladder cancer cell line KU-7 expressing GFP was from the Anticancer Inc. cell line bank. Transplanted animals were implanted by intravesical instillation using KU-7-GFP bladder cancer cells. Animals were anesthetized with a mixture of ketamine, acepromazine, and xylazine. The surgical area is disinfected with iodine and alcohol. After proper exposure of the bladder following a midline lower abdominal incision, the bladder is catheterized with a 24-G vascular catheter, drained and damaged by scoring with a needle in the bladder lining. KU-7-GFP (100. mu.l 2X 106) cells were instilled into the bladder and the purse-string ring was placed to seal the urethra to allow the cells to remain for 1 hour. The bladder is then returned to the abdominal cavity. The abdominal wall incision was closed in one layer with 6-0 surgical sutures. Maintaining animals in Isofluorfen during surgeryUnder alkane anesthesia. All procedures for the above operations were performed under a 7-fold magnification microscope (Olympus). Animals were kept in a barrier facility under HEPA filtration.

On day 7 post tumor cell implantation, fifty animals were randomly divided into five groups (each treatment group contained n ═ 10 mice) on day 7 post tumor implantation. Treatment of all groups of all mice began on the same day, which was considered to be day 0 of the study. Tables 13 and 14 show the study design. The 24G/3/4' IV catheter was used to instill fresh reconstituted formulation intravesically (50 μ L) and the urethra was knotted using a purse-string knot. The formulation was maintained in the bladder for 1 hour. After 1 hour, the peri-annular ring was dissected open to allow the bladder to empty naturally. The same procedure was followed on days 0, 7, 14 and 21.

As a result: animals treated with the proliposomal paclitaxel (PLIP) formulation showed a reduction in bladder tumor area compared to the saline group. The pure drug treated group lost six animals due to overexposure of the drug in solution (in DMSO), which could lead to systemic toxicity. Figure 8 shows the mean plasma levels showing minimal exposure of the drug to systemic circulation in the PLIP group, while pure drug dissolved in DMSO resulted in significant paclitaxel plasma levels, which is undesirable for the treatment of bladder cancer. Higher doses were studied and compared with commercial products based on lower doses

A comparison is made. PLIP formulations at 10mg/kg showed similar effects to Abraxane at 15 mg/kg. Increasing doses of PLIP formulations showed some reduction in tumor area (figures 5, 6 and 7).

TABLE 13 study design 1

Group of

Reagent

Dosage form

Plan for

Pathway(s)

n

1

PLIP-001

0.5mg/kg

Once a week for 4 weeks

Intravesical instillation

10

2

PLIP-001

2.5mg/kg

Once a week for 4 weeks

Intravesical instillation

10

3

PLIP-001

5mg/kg

Once a week for 4 weeks

Intravesical instillation

10

4

PTX in DMSO

5mg/kg

Once a week for 4 weeks

Intravesical instillation

10

5

Salt water

50μL

Once a week for 4 weeks

Intravesical instillation

10

TABLE 14 study design 2

Group of

Reagent

Dosage form

Plan for

Pathway(s)

n

1

PLIP-001

10mg/kg

Once a week for 2 weeks

Intravesical instillation

10

2

PLIP-001

15mg/kg

Once a week for 2 weeks

Intravesical instillation

10

3

Abraxane

15mg/kg

Once a week for 2 weeks

Intravesical instillation

10

4

Salt water

50 uL/mouse

Once a week for 2 weeks

Intravesical instillation

10

TABLE 15 study design 1 Final tumor area after four weeks of treatment

TABLE 16 study design 1 Final histology tumor area after four weeks of treatment

(in the table, a symbol denotes a tumor outside the bladder).

Table 17 study design 2 final tumor area measured by fluorescence method two weeks after treatment

Example 10 evaluation of metastases in mice of study design 2: group 1 (10mg/kg PLIP-001), group 2 (15mg/kg PLIP-001) and group 3 (15mg/kg Abraxane). Tables 18, 19 and 20 show the incidence of metastases in the following organs: liver, mesentery, diaphragm and kidney.

TABLE 18 metastases in group 1

TABLE 19 metastases in group 2

TABLE 20 metastases in group 3

TABLE 21 summary of intravesical treatment response in orthotopic nude mouse model

(EV as extra-vesical dilatation, i.e. tumor present outside the bladder)

Example 11 Paclitaxel (PTX) is highly active against metastatic bladder cancer, and thus PTX is a potential candidate for adjuvant intravesical therapy to prevent recurrence and progression of N-IBC. PTX is lipophilic. The existing formulations (e.g., paclitaxel/Abraxane) do not dissolve in the typical acidic aqueous intravesical environment. The lipophilic nature of PTX, if properly formulated, creates the potential for urothelial penetration and delivery into the submucosa. The purpose of this study was to demonstrate successful delivery of PTX (using liposomes) to the bladder, as well as in vitro and in vivo demonstration of the concept of PLIP.

IC50 values were evaluated using an in vitro human bladder cancer cell line (T24, KU 7). In vivo studies were performed in nude mice inoculated with KU7-GFP cell line. Following KU7 bladder tumor inoculation, weekly (x3) intravesical infusions (group 3: PLIP; PTX/DMSO or PTX/Nab; or saline) were performed and tumor growth was measured. Pharmacokinetic studies were performed in rat species. Acute up-scaling toxicology/pharmacokinetic studies of GLP compliance were also performed. Ex vivo porcine bladder model and PTX tissue concentration were compared (PLIP vs Abraxane).

Study No.1 results: the IC50 resistance to T24 human bladder cancer was 0.01 for PLIP and 0.5. mu.g/mL for the Abraxane PTX formulation. PLIP was effective in significantly reducing tumor size and improving the complete response rate to saline (figure 7/table 22). PLIP demonstrated a greatly reduced systemic exposure to PTX with mortality lower than PTX/DMSO. In an in vitro isolated porcine bladder model, plip (vs abraxane) allows for excellent transfer of paclitaxel from intravesical liposomes to the urothelium and urothelial subbing layers of the bladder without systemic exposure and associated toxicity. See fig. 9.

TABLE 22

(. in the table shows statistically significant p <0.05 difference compared to saline control)

These data confirm that PLIP is stable in human urine under in vitro conditions, highly active against human bladder tumor cell lines tested in vitro and in vivo, and delivers higher concentrations of PTX to urothelial tissue than Abraxane, with negligible systemic levels of PTX.

Example 12 in vitro adhesion/fusion/transport studies using porcine bladder.

Experiment: fresh porcine bladder was obtained from a slaughterhouse (n ═ 3) (male) and any remaining urine was drained. The excised bladders were washed with cold Kerb buffer. The excised bladders were then washed and stored in cold Tyrode buffer until the start of the experiment. The bladder was flushed through the urethra with 5mL of Tyrode buffer (37 ℃). Lyophilized PLIP and Abraxane formulations (6mg) were reconstituted with 5mL of Tyrode buffer (37 ℃). The formulation (5mL) was added to the bladder via the urethra. Immediately after addition, 0.5mL of the applied formulation was removed to evaluate the zero time (T0) sample. The bladder was then placed in 150mL Tyrode buffer (37 ℃) and placed in a water bath shaker for 2 hours. After 2 hours, the bladder contents were emptied and samples were collected for analysis. The bladder was flushed with 5mL of Tyrode buffer (37 ℃) and a sample was collected for analysis (this step was performed twice). The bladder was dissected and a small section (1-2 g in weight) was cut out. A piece of tissue was used for cryomicrotome sectioning. The cryomicrotome was performed at-15 ℃ and 10X 50 μm sections were collected in Eppendorf tubes for extraction. The sections were cut until the muscle layer (in which it was difficult to cut) was reached. Extraction of the sections or whole pieces was performed using methanol and analyzed using HPLC method for determination of the formulation. The results show that PLIP can penetrate the urinary epithelial layer and is able to deliver drugs better than Abraxane (figure 9). However, no drug levels were observed beyond 2500 μm in the urinary epithelium layer. The intrinsic layer depth was 2500 μm. This is an important inventive attribute, as the PLIP formulation delivers paclitaxel to the non-muscle anatomical limits of the bladder to prevent tumor growth, while not showing any systemic exposure to the drug.

Example 13 pharmacokinetic studies in female Sprague-Dawley rats. Evaluation of plasma PK profiles and PLIP versus bladder following a single intravesical administration in the bladder of female Sprague Dawley rats

Bladder concentration of (a). PLIP and Abraxane were administered once for 2 hours intravesical instillationThe infusion period was followed by a 24 hour post-dose (post-dose) observation period (Table 23).

TABLE 23 intravesical PK study design for female SD rats

Target dose is based on average body weight of about 0.300 kg/rat.

Animals were administered PLIP or under isoflurane anesthesia by using a urethral intravesical catheter followed by a 2 hour bladder retention period slow bolus into the bladder

The exposure time of 2 hours is based on technical feasibility and the maximum dose volume is calculated based on the urine volume of the rats. At the end of the dosing/retention period, the dosage formulation was expelled from the bladder by gently palpating the bladder through the abdominal wall. During this study, the assessments included mortality checks and clinical observations. Plasma samples for PK analysis were collected on day 1 at the following target time points: before dosing and 1,2, 3, 4, 6 and 24 hours after infusion has begun. At the end of 24 hours, the bladders were collected and snap frozen for analysis of paclitaxel concentration.

There was no PLIP-related effect on mortality or clinical observations. A single intravesical infusion of PLIP with a 2 hour retention time at a concentration of 3mg/mL (1.5 mg/animal) resulted in non-quantifiable plasma paclitaxel levels (lower limit of quantitation [ LLOQ ] in all treated animals]＝1ng/mL]). At the same dose (1.5 mg/animal)

Similar results were obtained in the comparative group, except that the concentrations in the two animals were 1.04ng/mL at 2.17 hours after the start of infusion and 1.76ng/mL at 3 hours after the start of infusion, respectively. These findings support the conclusion that: PLIP does not have systemic bioavailability when administered by intravesical route of administration at the maximum feasible dose in rats.

6 hours and 24 hours after the start of infusionThe results of the bladder tissue analysis showed that the results in PLIP or

Thereafter, paclitaxel is absorbed into the bladder; however, at 6 hours, the results varied within each treatment group. Paclitaxel concentration in bladder after 6 hours 1 and 4 of 4 PLIP-treated animals

3 of the treated animals were in the range of about 300 ng/g. In the PLIP group, one of all treated animals had the lowest bladder paclitaxel concentration (approximately 40ng/g), while two animals in this group had values in the range of approximately 1800-1900 ng/g. In that

In the treatment group, one animal had a bladder concentration of approximately 8500ng/g, while the remaining three animals were in the 300ng/g range. The reason for the variability of the data at 6 hours is not known, but may be related to the residual dose formulation remaining in the bladder after mechanical massage of the bladder to help neutralize the infusion. At 24 hours after infusion began, the bladder paclitaxel concentration was significantly lower than the 6 hour paclitaxel concentration, which might be expected from the urine flow to help remove residual dose formulation from the inner bladder surface and potentially metabolize or further distribute paclitaxel.

TABLE 24 in vivo bladder Taxol drug concentrations

Example 14 preparation of proliposomal intravesical cisplatin (PLIC) preparation PLIC-002.

PLIC-002 was prepared by dissolving 18.4mg of cisplatin in 15mL of water. The aqueous cisplatin solution was combined with 3ml of an ethanol solution containing the lipid components listed in table 25 at room temperature. Use of

-C5 homogenizer extrusion of the prepared dispersion. Extrusion was carried out using a polycarbonate film with a pore size decreasing from 1 μm to 0.2. mu.m. For final extrusion, 100mg of mannitol was mixed with the extrudate, and the mixture was lyophilized to obtain proliposomes.

TABLE 25PLIC-002.

Composition (I)	Measurement of
		Cisplatin (mw 300Da)	18.4mg
DMPG(mw＝688.9Da；Tc＝23℃)	22mg
		DMPC(mw＝677.9Da；Tc＝24℃)	51mg
Cholesterol (mw 386.65Da)	16mg
		Mannitol	100mg

Example 15 preparation of PLIC-009. PLCP-009 was prepared by dissolving 9.8mg of cisplatin in 11mL saline solution. The cisplatin aqueous solution was combined with 4ml of ethanol solution containing the lipid components in table 26 at room temperature. The prepared dispersion was then extruded using an emuisiflextm-C5 homogenizer. Extrusion was carried out using a polycarbonate film with a pore size decreasing from 1 μm to 0.2. mu.m. For the final extrusion, 26mg of mannitol was mixed with the extrudate, and the mixture was lyophilized to obtain proliposomes.

TABLE 26 PLIC-009.

Composition (I)	Measurement of
		Cis-platinum	9.8mg
DMPG	22.2mg
		DMPC	40.8mg
Mannitol	26mg

Example 16 in vitro analysis of the effectiveness of the Cisplatin (CPN) proliposomal preparation

Measurement of cisplatin preparations PLIC-002 and PLIC-009 on human bladder cancer epithelial cell line T24 by Sulforhodamine B (SB) -based assay method

HTB-4^TM)，5637(

HTB-9^TM) And HT-1376(

CRL-1472^TM) The inhibitory concentration IC 50. For use in the assay, cisplatin preparations were usedAnd then redispersed in normal saline to a concentration of 2mg/mL cisplatin. The redispersed formulation formed a clear solution. A1 mg/mL solution of pure cisplatin (unformulated) in physiological saline was used as a control. Higher concentrations of pure cisplatin were not used because cisplatin did not form clear solutions above 1mg/mL in physiological saline.

Cells were treated at 5X 10³Individual cells/well were plated onto 96-well plates at 37 ℃ and 5% CO₂And culturing for 24 hours. 2mg/mL cisplatin preparation and 1mg/mL pure drug control were added to the culture medium of the plate cell cultures at a dose of 100. mu.L. After 72h of treatment with the formulation, the medium was aspirated. The treated cells were then fixed by slowly adding 100 μ l of 10% trichloroacetic acid (TCA) to each well and the plates were incubated at 4 ℃ for at least 1 h. After incubation, the plates were washed five times with tap water without flowing water directly into the wells, the plates were air dried at room temperature, and 50 μ Ι of 0.4% w/v SRB (in 1% acetic acid) was added to each well. The plates were incubated in SRB solution for 20 to 30 minutes at room temperature. After that, the plate was washed 5 times with 1% acetic acid and air-dried at room temperature. Protein-bound SRB was detected by adding 100. mu.l of 10mM Tris base solution to each well and allowing the Tris solution to dissolve the SRB for 5 to 10 minutes. The plates were read using a microplate reader at 565nm absorbance. Table 27 reports the IC50 values for PLIC-002, PLIC-009, and pure drug solutions.

Watch 27

Preparation	Cell lines	IC50(μg/mL)
			PLIC-002	T24	1.283
PLIC-002	5637	0.692
			PLIC-002	HT1376	2.292
PLIC-009	T24	0.8658
			PLIC-009	5637	0.394
PLIC-009	HT1376	No test was made
			Pure medicine	T24	0.788
Pure medicine	5637	0.441
			Pure medicine	HT1376	1.11

Example 17 in vitro analysis of the effectiveness of docetaxel formulation (DTL-102716)

Watch 28

Composition (I)	Measurement of
		DMPC	8.6mg
DMPG	3.4mg
		Anhydrous docetaxel, USP	6mg
Mannitol	15mg
		Water (W)	1mL

Docetaxel formulations were prepared using the same method as described in example 6 above. The average particle size (Zave) in the formulation was 380 nm. The inhibitory concentration IC50 of docetaxel formulations on KU-7 cell line was determined using an in vitro sulforhodamine b (srb) -based assay as described above for paclitaxel. Docetaxel formulation had an IC50 of 0.0005 ng/mL.

Claims (15)

1. A proliposome powder dispersion comprising

(a) A taxane or a cisplatin, or a pharmaceutically acceptable salt thereof,

(b) dimyristoyl phosphatidylcholine (DMPC), and

(c) dimyristoyl phosphatidylglycerol sodium (DMPG),

wherein the weight ratio of a to b to c is 1.0 (1.3-4.5) to 0.4-2.5.

2. The proliposomal powder dispersion of claim 1, wherein (a) is a taxane selected from the group consisting of paclitaxel, docetaxel, cabazitaxel, tesetaxel, DJ-927, TPI 287, larotaxel, otaxel, DHA-paclitaxel, and mixtures thereof.

3. The proliposome powder dispersion of claim 1, wherein (a) is docetaxel and the weight ratio of a: b: c is 1.0 (1.3-2.0) to (0.4-2.0).

4. The proliposome powder dispersion of claim 1, wherein (a) is cisplatin and said dispersion further comprises (d) cholesterol, and the weight ratio of a: b: c: d is 1.0 (2.5-4.5): (1.0-2.5): (0.5-1.0).

5. A proliposome powder dispersion comprising

(a) The amount of paclitaxel is such that the amount of paclitaxel,

(b) dimyristoyl phosphatidylcholine (DMPC), and

(c) dimyristoyl phosphatidylglycerol sodium (DMPG),

wherein the weight ratio of a to b to c is 1 (1.3-3.8) to 0.4-1.5.

6. A proliposome powder dispersion according to claim 1 or 5, which additionally comprises (d) cholesterol,

wherein the weight ratio of a to b to c to d is 1.0 (1.3-3.8) to 0.4-1.5 to 0.5-1.0.

7. A pharmaceutical composition comprising the proliposomal powder dispersion of any one of claims 1-6 and at least one pharmaceutically acceptable excipient.

8. A method of preparing a liposomal formulation of taxane or cisplatin comprising hydrating a proliposomal powder dispersion of any one of claims 1-6 in an aqueous carrier.

9. A process for preparing a taxane or cisplatin formulation comprising the steps of:

(i) dispersing the first lipid and the second lipid in the aqueous carrier by stirring, mixing and/or homogenizing to form a dispersion;

(ii) adding a taxane or cisplatin to the dispersion of the first lipid and the second lipid;

(iii) homogenizing a dispersion of a first lipid, a second lipid and taxane or cisplatin to obtain taxane-or cisplatin-incorporated liposomes;

(iv) homogenizing the liposomes to obtain nanoscale liposome particles in dispersion; and

(v) cryoprotectants/lyoprotectants were added.

10. The method of claim 9, wherein the method further comprises the step of (vi) lyophilizing the dispersion to form a proliposome powder dispersion.

11. The process of claim 9, wherein step (iii) is carried out at elevated pressure and/or at a temperature above the Tc/Tg of the lipid.

12. A pharmaceutical composition comprising a liposomal formulation prepared by the process of claim 8 or 9 and at least one pharmaceutically acceptable excipient.

13. A method of treating bladder cancer in a patient, comprising the step of administering to the patient the pharmaceutical composition of claim 12, wherein the pharmaceutical composition is administered by intravesical delivery, and wherein the cancer is a non-muscle invasive bladder cancer.

14. The method of claim 13, wherein the taxane or cisplatin remains soluble in the bladder at any pH of 4.5-8.

15. A method of treating an upper urothelial cancer in a patient, comprising the step of administering to the patient the pharmaceutical composition of claim 12, wherein the pharmaceutical composition is administered into the ureter and/or renal pelvis.

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